Rna capping method, production method for modified rna, and modified rna

Abstract

An RNA capping method including a step of reacting a compound (1) represented by the following general formula (1) with an RNA in the presence of a Vaccinia virus capping enzyme (Rb1 represents an oxo group, an alkoxy group, or a halogen atom; Rb2 is either absent or represents an alkyl group; Rb3 represents an amino group or a hydrogen atom; Rb4 is either absent or represents a hydrogen atom or an alkyl group; Rr1 represents a hydroxy group, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR1C≡CH, or —R2R3; Rr2 represents a hydrogen atom, a hydroxy group, a halogen atom, an alkoxy group, an amino group, an azide group, —OR1C≡CH, or —R2R3; and A1 represents an oxygen atom or a sulfur atom).

1 . An RNA capping method comprising
 reacting a compound (1) represented by the following general formula (1), provided that GTP is excluded, with an RNA in the presence of a Vaccinia virus capping enzyme,   
       
         
           
           
               
               
           
         
         wherein R b1  represents an oxo group, an alkoxy group having 1 to 3 carbon atoms, or a halogen atom; R b2  is either absent or represents an alkyl group having 1 to 3 carbon atoms; R b3  represents an amino group or a hydrogen atom; R b4  is either absent or represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R r1  represents a hydroxy group, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR 1 C≡CH, or —R 2 R 3 ; R r2  represents a hydrogen atom, a hydroxy group, a halogen atom, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR 1 C≡CH, or —R 2 R 3 ; A 1  represents an oxygen atom or a sulfur atom; 
         R 1  represents an alkylene group having 1 to 3 carbon atoms, R 2  represents —OC(═O)NH—, —OC(═O)—, —NHC(═O)—, —O— or a single bond; and R 3  represents an aminoalkyl group having 1 to 3 carbon atoms; and 
         a double line composed of a solid line and a dotted line represents a single bond or a double bond. 
       
     
     
         2 . The RNA capping method according to  claim 1 ,
 wherein the compound (1) is selected from the group consisting of N7-methylguanosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, 6-chloroguanosine-5′-triphosphate, inosine-5′-triphosphate, 2′-deoxyguanosine-5′-triphosphate, araguanosine-5′-triphosphate, 2′-fluoro-2′-deoxyguanosine-5′-triphosphate, 2′-O-methylguanosine-5′-triphosphate, 3′-O-methylguanosine-5′-triphosphate, 2′-amino-2′-deoxyguanosine-5′-triphosphate, 3′-amino-2′,3′-dideoxyguanosine-5′-triphosphate, 2′-azido-2′-deoxyguanosine-5′-triphosphate, 3′-azido-2′,3′-dideoxyguanosine-5′-triphosphate, 3′-(O-propargyl)-guanosine-5′-triphosphate, 2′/3′-O-(2-aminoethyl-carbamoyl)-guanosine-5′-triphosphate and guanosine-5′-O-(1-thiotriphosphate).   
     
     
         3 . A method for producing a modified RNA, comprising (a) reacting a compound (1) represented by the following general formula (1), provided that GTP is excluded, with an RNA in the presence of a Vaccinia virus capping enzyme, 
       
         
           
           
               
               
           
         
         wherein R b1  represents an oxo group, an alkoxy group having 1 to 3 carbon atoms, or a halogen atom; R b2  is either absent or represents an alkyl group having 1 to 3 carbon atoms; R b3  represents an amino group or a hydrogen atom; R b4  is either absent or represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R r1  represents a hydroxy group, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR 1 C≡CH, or —R 2 R 3 ; R r2  represents a hydrogen atom, a hydroxy group, a halogen atom, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR 1 C≡CH, or —R 2 R 3 ; A 1  represents an oxygen atom or a sulfur atom; 
         R 1  represents an alkylene group having 1 to 3 carbon atoms, R 2  represents —OC(═O)NH—, —OC(═O)—, —NHC(═O)—, —O— or a single bond; and R 3  represents an aminoalkyl group having 1 to 3 carbon atoms; and 
         a double line composed of a solid line and a dotted line represents a single bond or a double bond. 
       
     
     
         4 . The method for producing a modified RNA according to  claim 3 ,
 wherein the compound (1) is selected from the group consisting of N7-methylguanosine-5′-triphosphate, N 1 -methylguanosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, 6-chloroguanosine-5′-triphosphate, inosine-5′-triphosphate, 2′-deoxyguanosine-5′-triphosphate, araguanosine-5′-triphosphate, 2′-fluoro-2′-deoxyguanosine-5′-triphosphate, 2′-O-methylguanosine-5′-triphosphate, 3′-O-methylguanosine-5′-triphosphate, 2′-amino-2′-deoxyguanosine-5′-triphosphate, 3′-amino-2′,3′-dideoxyguanosine-5′-triphosphate, 2′-azido-2′-deoxyguanosine-5′-triphosphate, 3′-azido-2′,3′-dideoxyguanosine-5′-triphosphate, 3′-(O-propargyl)-guanosine-5′-triphosphate, 2′/3′-O-(2-aminoethyl-carbamoyl)-guanosine-5′-triphosphate and guanosine-5′-O-(1-thiotriphosphate).   
     
     
         5 . The method for producing a modified RNA according to  claim 3 , further comprising, after (a),
 (b1) reacting an alkyne compound,   wherein R r1  or R r2  in the general formula (1) is an azide group.   
     
     
         6 . The method for producing a modified RNA according to  claim 5 , wherein the compound (1) is 2′-azido-2′-deoxyguanosine-5′-triphosphate or 3′-azido-2′,3′-dideoxyguanosine-5′-triphosphate. 
     
     
         7 . The method for producing a modified RNA according to  claim 5 , wherein the alkyne compound comprises a functional group. 
     
     
         8 . The method for producing a modified RNA according to  claim 3 , further comprising, after (a),
 (b2) reacting an azide compound,   wherein R r1  or R r2  in the general formula (1) is —OR 1 C≡CH.   
     
     
         9 . The method for producing a modified RNA according to  claim 8 , wherein the compound (1) is 3′-(O-propargyl)-guanosine-5′-triphosphate. 
     
     
         10 . The method for producing a modified RNA according to  claim 8 , wherein the azide compound comprises a functional group. 
     
     
         11 . The method for producing a modified RNA according to  claim 3 , further comprising
 (c1) binding a compound (Ak-1) represented by the following general formula (Ak-1) to a 3′ end of the RNA,   wherein R r1  or R r2  in the general formula (1) is an azide group,   
       
         
           
           
               
               
           
         
         wherein Y 1  represents a divalent linking group; and Base represents a nucleotide base. 
       
     
     
         12 . The method for producing a modified RNA according to  claim 11 , further comprising, after (a) and (c1),
 (d1) producing a circular RNA by reacting an azide group introduced into a 5′ end of the RNA by (a) with an alkynyl group introduced into a 3′ end of the RNA by (c1).   
     
     
         13 . The method for producing a modified RNA according to  claim 11 , wherein the compound (1) is 2′-azido-2′-deoxyguanosine-5′-triphosphate or 3′-azido-2′,3′-dideoxyguanosine-5′-triphosphate. 
     
     
         14 . The method for producing a modified RNA according to  claim 3 , further comprising
 (c2) binding a compound (Az-1) represented by the following general formula (Az-1) to a 3′ end of the RNA,   wherein R r1  or R r2  in the general formula (1) is —OR 1 C≡CH,   
       
         
           
           
               
               
           
         
         wherein Y 2  represents a divalent linking group; and Base represents a nucleotide base. 
       
     
     
         15 . The method for producing a modified RNA according to  claim 14 , further comprising, after (a) and (c2),
 (d2) producing a circular RNA by reacting an alkynyl group introduced into a 5′ end of the RNA by (a) with an azide group introduced into a 3′ end of the RNA by (c2).   
     
     
         16 . The method for producing a modified RNA according to  claim 14 , wherein the compound (1) is 3′-(O-propargyl)-guanosine-5′-triphosphate. 
     
     
         17 . A modified RNA, which is a modified RNA comprising a structure selected from the group consisting of the following formulas (Cp-1) to (Cp-13) and (Ca-1) to (Ca-3) at a 5′ end, or a circular modified RNA comprising a structure selected from the group consisting of the following formulas (Ci-1) to (Ci-3), 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein m 7 G represents N7-methylguanine; * represents a bond that binds to a carbon atom at a 5′ position of an adjacent nucleotide residue; 
         R a1  and R a2  each independently represent a hydrogen atom or an organic group, provided that at least one of R a1  and R a2  is an organic group, 
         when both R a1  and R a2  are organic groups, they may bind with each other to form a ring structure, 
         R a3  represents an organic group; 
         Y 1  and Y 2  each independently represent a divalent linking group; Base each independently represents a nucleotide base; and ** represents a bond that binds to a carbon atom at a 3′ position of an adjacent nucleotide residue. 
       
     
     
         18 . (canceled) 
     
     
         19 . (canceled)